The Biophysics of Developmentally Regulated Gene Transcription

基因转录的启动可以说是多细胞生物体发育过程中最有影响力的控制点。在基因激活的最简单版本中，特定的转录因子与目标基因控制区的短DNA序列(6-12bps)结合，然后以某种方式招募RNA聚合酶II及其辅助蛋白的巨大生化机制，从而启动转录。所有这些都发生在“染色质”的环境中，以可能的辅因子、差异修饰的组蛋白和潜在定位的核小体为特征。我们将集中在非常重要的基因激活的早期步骤，在这一步骤中，单个控制转录因子与其在目标基因调节区的结合部位相互作用。我们的目标是将体外用纯化成分进行的精确生物物理测量与体内测量的定量转录结果联系起来。 多年来，我们一直在分析和描述一种简单的克隆细胞谱系--小型自由生活线虫秀丽线虫的肠道。所有的主要转录因子都是已知的，但我们将重点关注ELT-2，它是从早期胚胎到死亡为止推动肠道分化的主要转录因子。我们建议通过实验来定义ELT-2与其在胚胎内的结合位置之间相互作用的三个基本方面。 目的1：ELT-2优先与TGATAA DNA结合，但不同的侧翼序列导致不同的结合亲和力。利用先进的显微技术，我们将测试ELT-2在体外和体内，即在活胚胎的细胞核内，是否具有与DNA结合的相同光谱。我们还将测量活胚胎中游离ELT-2蛋白的水平。这两个结果对于在定量生物物理水平上理解基因调控是必要的。 目的2：我们开发了一个严格控制的实验系统，在该系统中，ELT-2结合位点的单碱基变体在动物体内具有显著的转录后果。我们将首先定义ELT-2亲和力和转录水平之间的关系。然后将该系统扩展到测量发育阶段、染色质蛋白和染色质修饰的影响。我们将提出一个关键问题：其他靶基因是否也遵循同样的规则？ 目的3：我们将结合对线虫肠道的低技术解剖和可获得DNA的最新标记，从而确定核小体在肠道特异基因调控区域中的位置。 这项研究的目的是在结合能的基础水平上了解胚胎发育中关键决定是如何做出的。由于ELT-2与驱动脊椎动物内胚层发育的转录因子GATA-4/5/6同源，这一发现可能适用于所有动物的肠道发育，包括我们自己。

Initiation of gene transcription is arguably the most influential control point in the development of a multicellular organism. In the simplest version of gene activation, a specific transcription factor binds to a short DNA sequence (6-12 bps) in the control region of a target gene and then somehow recruits the vast biochemical machinery of RNA Polymerase II and its auxiliary proteins, thereby initiating transcription. All of this occurs in an environment of “chromatin”, featuring possible cofactors, differentially modified histones and potentially positioned nucleosomes. We will focus on the all-important early step of gene activation in which a single controlling transcription factor interacts with its binding site in the regulatory region of a target gene. Our goal is to relate precise biophysical measurements made with purified components in vitro to quantitative transcriptional consequences measured in vivo. For years, we have been analyzing and describing the development of a simple clonal cell lineage, the intestine of the small free-living nematode worm Caenorhabditis elegans. All the major transcription factors are now known but we will focus on ELT-2, the predominant transcription factor driving intestinal differentiation from the early embryo until death. We propose experiments to define three fundamental aspects of the interaction between ELT-2 and its binding sites inside an embryo. Aim 1: ELT-2 binds preferentially to TGATAA DNA sites but different flanking sequences cause different binding affinities. Using advanced microscopic techniques, we will test if ELT-2 has the same spectrum of affinities binding to DNA in vitro and in vivo, i.e. inside the nucleus of a living embryo. We will also measure the level of free ELT-2 protein inside a living embryo. Both results are necessary to understand gene regulation at a quantitative biophysical level. Aim 2: We have developed a tightly controlled experimental system in which single base pair variants of the ELT-2 binding site have significant transcriptional consequences inside the animal. We will first define the relation between ELT-2 affinity and transcript levels. and then extend this system to measure influences of developmental stage, chromatin proteins and chromatin modifications. We will ask the key question: do other target genes follow the same rules? Aim 3: We will combine “low-tech” dissection of C. elegans intestines with state-of-the-art tagging of accessible DNA, thereby defining the position of nucleosomes in regulatory regions of intestine specific genes. The aim of this research is to understand, at the fundamental level of binding energies, how critical decisions are made in embryonic development. Because ELT-2 is homologous to transcription factors GATA-4/5/6 driving endoderm development in vertebrates, the findings are likely to apply to development of intestines in all animals, including ourselves.